Flaviviruses are a major cause of infectious disease in human, which include the Dengue Virus (DENV), West Nile virus (WNV), Japanese encephalitis virus (JEV) and yellow fever virus (YFV). The genomic RNA encodes a polyprotein precursor which is processed proteolytically upon translation to 10 proteins, including three structural proteins (capsid [C], premembrane [prM] and envelope [Env]), and seven nonstructural (NS) proteins (NS1, NS2A/B, NS3, NS4A/B, and NS5). The NS3 is of great intereste in drug discovery because of its protease activity required for viral replication. The activity of NS3 is regulated by NS2B that is a membrane protein. Previous studies were using a construct that contains 40 amino acids from NS2B fused with NS3 protease domain though an artificial G4SG4 linker. We developed a series of peptidic inhibitors targeting WNV protease. We analyzed their interactions with the protease using both chemical shift perturbation and docking studies. For the DENV protease, there is still no potent inhibitor available so far. Our recent study suggested that the conventional DENV NS3 protease construct in which NS2B cofactor region linked with NS3 protease through a flexible liner is not suitable for drug discovery due to the protein dynamics. Using a co-expression system, we obtained a protease complex that contains the NS3 protease domain and 50-residue segment of the NS2B. Our results show that this protease complex exists as a close conformation and active under physiological conditions, which might be a better construct for drug discovery targeting DENV.